The primary structure of non-naturally-occurring polymeric materials—that is, the sequential arrangement of monomer units in a polymer chain—is generally poorly controlled in synthetic macromolecules. Common non-natural polymers are usually homopolymers, made of the same monomer unit, or copolymers with simple chain microstructures, such as random or block copolymers. These polymers are used in many areas but do not have the structural and functional complexity of defined sequence biopolymers, such as oligonucleotides, nucleic acids, proteins peptides, or oligosaccharides.
There is great utility in defined monomer sequence non-naturally-occurring polymers, i.e. non-biological polymers which are assembled from a library of functional building blocks so that the monomer order is exactly defined, and in which at least two or more of the monomers are structurally distinct from each other. For such molecules it may be possible to programme their structural properties, for example folding and self-assembly, and also their macroscopic properties (Lutz J-F et al., “Sequence-Controlled Polymers”, Science 9 Aug. 2013, Vol 341, page 628.) Many applications in medicine are also envisaged (Hartmann L and Borner H G, “Precision Polymers: Monodisperse, Monomer-Sequence-Defined Segments to Target Future Demands of Polymers in Medicine” Advanced Materials. 2009, Vol 21, pp 3425-3431).
A key challenge for defined monomer sequence polymers formed from non-naturally-occurring monomers is how to prepare them. Various strategies have been proposed, including biological methods and chemical synthesis using iterative steps in which the monomers are attached one-by-one in a given order. This method suffers from the difficulties of purification at each step. This challenge has been addressed to date (Lutz J-F et al., “Sequence-Controlled Polymers”, Science 9 Aug. 2013, Vol 341, page 628. and Hartmann L and Borner H G, “Precision Polymers: Monodisperse, Monomer-Sequence-Defined Segments to Target Future Demands of Polymers in Medicine” Advanced Materials. 2009, Vol 21, pp 3425-3431) through either advanced polymerisation chemistry or solid phase synthesis as used for sequence defined biopolymers, such as oligonucleotides and peptides.
Membrane processes are well known in the art of separation science, and can be applied to a range of separations of species of varying molecular weights in liquid and gas phases (see for example “Membrane Technology” in Kirk Othmer Encyclopaedia of Chemical Technology, 4th Edition 1993, Vol 16, pages 135-193). Nanofiltration is a membrane process utilising membranes whose pores are in the range 0.5-5 nm, and which have molecular weight (MW) cut-offs in the range of 200-3,000 Daltons. Nanofiltration has been widely applied to filtration of aqueous fluids, but due to a lack of suitable solvent stable membranes has not been widely applied to separation of solutes in organic solvents. Ultrafiltration membranes typically have MW cut-offs in the range 3,000 to 1,000,000 Daltons. Recently new classes of membranes have been developed which are stable in even the most difficult solvents as reported in P. Vandezande, L. E. M. Gevers and I. F. J. Vankelecom Chem. Soc. Rev., (2008), Vol 37, pages 365-405, some of which may be suitable for Organic Solvent Nanofiltration (OSN). Such membranes may be polymeric membranes, ceramic membranes, or mixed inorganic/organic membranes.
Membrane processes have been combined with chemical synthesis for the production of sequence defined biopolymers such as peptides and oligonucelotides. The use of membranes during peptide synthesis to separate growing peptides from excess reagents and reaction by-products was reported in U.S. Pat. No. 3,772,264. Peptides were synthesised in a liquid phase, with poly(ethylene glycol) (PEG) as a molecular anchoring group, and separation of the growing peptide chain from impurities was achieved with aqueous phase ultrafiltration. The separation required evaporation of the organic solvent after each coupling step, neutralisation followed by evaporation after each deprotection, and then for either coupling or deprotection, water uptake before ultrafiltration from an aqueous solution. Water was then removed by evaporation and/or azeotropic distillation before re-dissolving the PEG anchored peptide back into organic solvent for the next coupling or deprotection step.
U.S. Pat. No. 8,664,357 reports the use of organic solvent nanofiltration membranes in a process for preparing biopolymers selected from oligonucleotides, peptides and peptide nucleic acids.
US Patent Application US 20130072691 A1 describes the use of organic solvent nanofiltration membranes in the preparation of monodisperse (i.e. of similar or equal molecular weight), heterobifunctional (having a different functional group at either end of the polymer) synthetic polymers based on polyethylene glycol.
Research to date has focused on the provision of biopolymers (such as oligonucleotides, peptides and peptide nucleic acids) having a defined sequence of monomeric units. Given their widespread applicability, there remains a need for a process for preparing non-naturally-occurring defined monomer sequence polymers.
The present invention was devised with the foregoing in mind.